This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major goal of the Rossie laboratory is to identify the regulators and substrates of Ser/Thr protein phosphatase 5 (PP5), for which only a few have been identified to date. One important pathway in which PP5 has been implicated is the DNA damage response pathway. This pathway is essential for preventing the propagation of cells with damaged DNA, which can ultimately lead to cancer. Two non-redundant, functionally overlapping and closely related protein kinases, namely ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3 related) are activated in response to various forms of genotoxic stress. Once activated, ATM and ATR kinases phosphorylate their substrate proteins which lead to the specific cellular response pathways mediating either cell cycle arrest and DNA repair, or apoptosis. Loss-of-function mutations in both alleles of the human ATM gene gives rise to an autosomal recessive disease called ataxia telangiectasia (A-T). Patients suffering from A-T disorder risk predisposition to cancer and also other neuronal and immune dysfunctions and premature aging. ATR is activated in response to a variety of DNA damage and replication stress pathways. Deletion of the ATR gene leads to embryonic lethality in mice indicating that this gene is essential during development or for viability. Recently, PP5 has been shown to be essential for the activation of both these kinases (ATM and ATR), but the role of PP5 in controlling these kinases is not known. The goal of this study is to identify the potential substrates and/or targets of PP5 during activation of the DNA damage pathway using whole cell phosphoproteomics, which requires the powerful high-throughput mass spectrometry and bioinformatics capabilities that are available at the Center at Pacific Northwest National Laboratory. To do this we will compare the phosphoproteome of cells with different levels of PP5 activity and subjected to treatment with a DNA damaging agent. Two different strategies will be used to alter PP5 activity in cells. Specific Aims: 1. To compare and identify phosphoprotein targets in cells over-expressing wild type (WT) and catalytically inactive mutant PP5 following DNA damage. 2. To compare and identify phosphoprotein targets specific for PP5 from cells over-expressing WT or mutant PP5 (insensitive to okadaic acid) following treatment with a DNA damaging agent in the absence or presence of okadaic acid (at a dose that blocks WT but not mutant PP5). Methods for Specific Aim 1: Stable human HeLa cell lines over-expressing either wild type (WT) or catalytically inactive (CaIn) PP5 under inducible conditions will be used for this study. Cells induced to over-express PP5 will be treated with or without bleomycin to activate the DNA damage response pathway. Protein extracts will be prepared using the Trizol method and digested with trypsin. Peptides will be carboxymethylated and phosphopeptides will be isolated by immobilized metal-ion affinity chromatography (IMAC), and then subjected to electrospray ionization interfaced with mass spectrometry. Differential isotopic labeling, either at the level of amino acid incorporation during cell growth (SILAC) or during carboxymethylation will be used to distinguish peptides arising from the samples to be compared. Once samples have been differentially labeled, peptides can be mixed prior to IMAC and LC-MS analysis. We predict that phosphopeptides from proteins targeted by WT PP5 during activation of the DNA damage pathway will be lower in abundance than their counterparts from cells expressing CaIn PP5. In addition to PP5 substrates, we expect to identify proteins whose phosphorylation status is indirectly altered as a function of PP5 activity during activation of this pathway. Based on the mass spectrometry data, further biochemical characterization of putative targets will be performed. If successful with confirmatory results from biochemical characterizations, these data will be published. Methods for Specific Aim 2: Stable human HeLa cell lines over-expressing either WT PP5 or okadaic acid (OA) insensitive mutant PP5 [OA-insensitive] under inducible conditions will be used for this study. OA is a Ser/Thr phosphatase inhibitor. Cells induced to over-express PP5 will be treated with or without OA. Following OA treatment, cells will be treated with or without DNA damaging agent bleomycin. OA treatment will inhibit WT PP5 and all other related Ser/Thr phosphatases in cells over-expressing WT PP5. However, in the cells over-expressing the OA-insensitive mutant PP5, the same Ser/Thr phosphatases, except for mutant PP5, will be inhibited and thus any changes in phosphoproteins in mutant PP5 over-expressing cells compared to the WT PP5 over-expressing cells can be correlated to PP5 activity. After OA treatment followed by DNA damage, whole cell extracts will be prepared and processed using mass spectrometry as mentioned in Specific Aim 1. In contrast to Specific Aim 1 in which long term effects of altering PP5 function may be observed, this experiment can better document the rapid changes in phosphorylation mediated by PP5, because the difference in PP5 function between samples lasts only for the duration of the experiment. Follow-up experiments testing the relationship between PP5 and altered phosphoproteins (identified in this study) are expected to lead to additional publications.